SUMMARY Tuberculosis (TB) remains a major global health burden with 9.6 million new cases in 2014 and a latently- infected population of billions and deaths from TB now exceed those from HIV. Mtb is a sophisticated pathogen which can persist for decades in the human host and which requires lengthy treatment for cure. One of the features of Mtb is its ability to survive and replicate inside human cells, including macrophages, one of the normal host defense mechanisms against infection. There is an urgent need for new drugs for TB and new drug targets for Mtb. An increased effort in drug discovery has led to the development and application of screening technologies to Mtb; in particular screening against axenically-cultured bacteria. However, laboratory medium and culture conditions do not accurately reproduce the in vivo setting. High-content analysis (HCA) is a powerful screening methodology which uses biologically relevant cell-based assays to identify active compounds in a high throughput manner. For intracellular pathogens, high content screening has the advantage of being able to monitor both bacterial and macrophage cell numbers simultaneously and in the same wells, thus leading to more reliable data and a quicker assessment of compound attractiveness. This proposal addresses NIH announcement PAR-13-364 - Development of Assays for High-Throughput Screening for Use in Probe and Pre-therapeutic Discovery (R01). We propose to develop and run robust and reproducible high content assay(s) for screening against intracellular Mtb. We will run a pilot screen to identify inhibitors which are differentially effective against intracellular bacteria, but which lack cytotoxicity, and so may target novel pathways relevant to infection. We will conduct orthogonal and secondary assays to prioritize compounds for the dual aspects of drug discovery and development, and as chemical probes and initiate target identification studies. This proposal will take advantage of a new facility recently established at our institute with the capability to conduct high throughput, high content screening under BSL3 using state of the art imaging equipment and robotics.